Assay Kit Name:
Citrate Assay Kit
• Detection method- Absorbance (570 nm) or Fluorescence (Ex/Em 535/587 nm)
• Sample type- Cell and Tissue culture supernatants, urine, as well as many other biological fluids
• Species reactivity- Mammalian
• Applications- The Citrate Assay Kit can detect 0.1 to 10 nmoles (~2 µM-10 mM) of citrate in a variety of samples.
Features & Benefits:
• Simple procedure; takes ~ 40 minutes
• Fast and convenient
• Kit contains all necessary reagents for accurate measurement of citrate levels
• Citrate Assay Buffer
• Citrate Probe (in DMSO)
• Citrate Enzyme Mix
• Citrate Developer
• Citrate Standard (10 µmol)
Citric acid (HOOC-CH₂-C(-OH)(-COOH)-CH₂-COOH) is a key intermediate in the TCA cycle which occurs in mitochondria. It is formed by the addition of oxaloacetate to the acetyl group of acetyl-CoA derived from the glycolytic pathway. Citrate can be transported out of mitochondria and converted back to acetyl CoA for fatty acid synthesis. Citrate is an allosteric modulator of both fatty acid synthesis (acetyl-CoA carboxylase) and glycolysis (phospho- fructokinase). Citrate is widely used industrially in foods, beverages and pharmaceuticals. Citrate metabolism and disposition can vary widely due to sex, age and a variety of other factors. BioVision's Citrate Assay Kit provides a simple, sensitive and rapid means of quantifying citrate in a variety of samples. In the assay, citrate is converted to pyruvate via oxaloacetate. The pyruvate is quantified by converting a nearly colorless probe to an intensely colored (570 nm) and fluorescent (Ex/Em, 535/587 nm) product. The Citrate Assay Kit can detect 0.1 to 10 nmoles (~2 µM-10 mM) of citrate in a variety of samples.
USAGE: For Research Use Only! Not For Use in Humans.
There is a lot of variation from one std curve to another with the same kit. Will preparing a master mix for the reactions help minimize this? Do you recommend pipetting up and down or using plate shaker?
We definitely recommend preparing master mixes for both reaction mix and background controls. This minimizes chances of pipetting errors from pipetting small volumes for each sample. We recommend using a plate shaker but shake gently so that there is no mixing between wells. Pipetting up/down is also possible but extreme care needs to taken to ensure no croos-contamination between wells.
How can cell culture media be used in this assay?
Cell culture medium can be directly used for detection of citrate with this kit. It is recommended to spin down for 1-2 minutes at top speed on a microcentrifuge to ensure there are no debris/cells floating in the medium. The volume of culture medium used per assay will need to be optimized.
Starting with 1 million cells what sample dilution is recommended for thie assay?
The optimum dilution factor depends on the cell type, the concentration of citrate in the cells and the overall quality of the lysate. I would recommend making a series of dilutions over a broad range (the datasheet recommends 1-50 ul per well) to make sure you get the best results which would read within the linear range of the standard curve.
What is the detection range of the assay? Can sodium citrate be detected by this kit?
This kit can detect 0.1 to 10 nmoles (~2 µM-10 mM) of citrate in a variety of samples. It can detect sodium citrate.
Can some the components be used beyond 2 months or they degrade by then?
The probe and all the reconstituted lyophilized components can be stored in aliquots at-20C. if never frozen and thawed, it is possible that the components will still work beyond 2 months. It is best to use within 2 months of reconstitution for best results.
What could be some of the incompatible sample types?
Incompatible samples can be any sample like blood collected with citrate as the anti-coagulant or medium with added citrate in it. This will likely cause saturation and skew the results due to the citrate concentrations being greater than the range (2uM-10mM).
Can individual components of this kit be purchased separately?
Yes, any of the kits components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can frozen samples be used with this assay?
Fresh samples are always preferred over frozen samples since this is an enzyme activity assay. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Is it essential to make a standard curve for every expt, or is one curve/kit enough?
Yes, it is strongly recommended to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.